The Extracellular Matrix Vitalizer RATM Increased Skin Elasticity by Modulating Mitochondrial Function in Aged Animal Skin

Oxidative stress-induced cellular senescence and mitochondrial dysfunction result in skin aging by increasing ECM levels-degrading proteins such as MMPs, and decreasing collagen synthesis. MMPs also destroy the basement membrane, which is involved in skin elasticity. The extracellular matrix vitalizer RATM (RA) contains various antioxidants and sodium hyaluronate, which lead to skin rejuvenation. We evaluated whether RA decreases oxidative stress and mitochondrial dysfunction, eventually increasing skin elasticity in aged animals. Oxidative stress was assessed by assaying NADPH oxidase activity, which is involved in ROS generation, and the expression of SOD, which removes ROS. NADPH oxidase activity was increased in aged skin and decreased by RA injection. SOD expression was decreased in aged skin and increased by RA injection. Damage to mitochondrial DNA and mitochondrial fusion markers was increased in aged skin and decreased by RA. The levels of mitochondrial biogenesis markers and fission markers were decreased in aged skin and increased by RA. The levels of NF-κB/AP-1 and MMP1/2/3/9 were increased in aged skin and decreased by RA. The levels of TGF-β, CTGF, and collagen I/III were decreased in aged skin and increased by RA. The expression of laminin and nidogen and basement membrane density were decreased in aged skin and increased by RA. RA increased collagen fiber accumulation and elasticity in aged skin. In conclusion, RA improves skin rejuvenation by decreasing oxidative stress and mitochondrial dysfunction in aged skin.


Introduction
The primary triggering factor of skin aging is oxidative cellular damage caused by increased oxidative stress [1,2]. Oxidative stress results from an imbalance between reactive oxygen species (ROS) synthesis and defense mechanisms that remove ROS [1,2]. Enzymes that remove ROS, such as glutathione (GSH), superoxide dismutase (SOD), and catalase, are representative of defense mechanisms against oxidative stress [3].
During the skin aging process, oxidative stress leads to the upregulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and its downstream signal which eventually decreased ECM fiber destruction and BM destruction. RA also increased the levels of TGF-β and CTGF, which eventually increased collagen fiber synthesis. We evaluated the RA-mediated increase in collagen fiber accumulation and decrease BM destruction via decreased oxidative stress in aged animals. The effects of RA were compared with those of AA or NA injected alone into aged animal's skin.

Preparation of RA
RA was formulated as a liquid before application. First, AA, NA, coenzymes, glutathione, and sodium hyaluronate were dissolved in distilled water with mixing at 3000 rpm using a high-speed mixer (T.K. Homo Disper, Model 2.5, PRIMIX, Hyogo, Japan). Then, the RA solution was filtered through a 0.2 µm filter (S2GPU11RE, Merck, Darmstadt, Land Hessen, Germany) to remove bacteria. The RA liquid contained 0.25% AA and 0.25% NA (Table S1).

Cell Culture
Human primary epidermal keratinocytes (HEKn; ATCC, Manassas, VA, USA) were cultivated with dermal cell basal medium (ATCC) with a keratinocyte growth kit (ATCC) and maintained at 37 • C under 5% CO 2 .

NA, AA, and RA Treatment
To determine whether keratinocytes were affected by NA, AA, or RA treatment, HEKn cells were treated with 50 µM H 2 O 2 for 2 h, treated with NA (0.4 mM), AA (0.4 mM), or RA (80 µL), and cultured for 48 h ( Figure S1A). In addition, control cells were treated with phosphate-buffered saline (PBS).

Mouse Conditions
Eight-week-old male C57BL/6 mice were obtained from Orient Bio (Seongnam, Republic of Korea). The young group contained 9-week-old mice after one week of acclimatization, and the aging group was bred until 12 months old.
This study was approved by the ethical board of the Center for Animal Care and Use. It was conducted by the guidelines of the Institutional Animal Care and Use Committee of Gachon University (approval number: LCDI-2022-0095). The mice used in this study were domesticated in an area with controlled temperature (22 ± 5 • C), relative humidity (50 ± 10%), and a 12-h light-dark cycle. In addition, they had free access to standard laboratory diets and water.

RA Treatment
To determine whether aged animal skin was affected by RA treatment, 12-month-old aging mice were injected intradermally with RA (100 µL/cm 2 /day) twice every two weeks using a microneedle therapy system (MTS; Derma-Q Gold 0.5 mm, DONGBANG medicare, Seongnam, Republic of Korea). The control was injected with distilled water under the same conditions ( Figure S1B).

Skin Elasticity
To confirm whether the skin elasticity of aged animal skin was changed by RA treatment, the skin elasticity of the animal before RA treatment and after 4 weeks was measured. Skin elasticity was evaluated with API-100 ® (Aram Huvis Co., Ltd., Seongnam, Republic of Korea), and the average was used after measuring 5 times for each animal.

Protein Isolation
Proteins were isolated from the cells and skin tissues by using the EzRIPA lysis kit (ATTO Corporation, Tokyo, Japan). First, the cells and skin tissues were lysed with EzRIPA buffer containing protease and phosphatase inhibitors. Then, the lysed samples were sonicated and centrifuged at 14,000× g for 20 min at 4 • C. Then, the supernatants were transferred to a new tube, and the protein was quantified by using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

RNA Extraction and cDNA Synthesis
The total RNA from cells and frozen skin tissues was extracted using RNAiso Plus (Takara Bio, Kusatsu, Japan) according to the manufacturer's instructions. The quality and concentration of the extracted RNA were confirmed by a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific), and cDNA was synthesized by using a PrimeScript First Strand cDNA Synthesis Kit (Takara Bio) according to the manufacturer's instructions.

Paraffin-Embedded Tissue
The harvested skin tissues were fixed with cold 4% paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS at 4 • C for 24 h. The fixed skin tissues were washed for 30 min, and a paraffin block was made using a tissue processor (Thermo Fisher Scientific). The paraffin blocks were sectioned to 7 µm in thickness using a microtome (Leica Biosystems, Nussloch, Germany) and dried at 60 • C overnight to allow them to attach to the slides.

Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase and SOD Activity
NADPH oxidase (Abcam, Cambridge, UK) and SOD (Abcam) activities in the H 2 O 2treated HEKn cells and skin tissue of each group were determined by using appropriate kits, following the manufacturers' instructions.

Enzyme-Linked Immunosorbent Assay (ELISA)
To measure the levels of the 8-hydroxy-2 -deoxyguanosine (8-OHdG), Collagen type I alpha 1 (COL1A1), and Collagen type III alpha 1 (COL3A1), 96-well microplates were coated in 100 nM carbonate and bicarbonate-mixed buffer, adjusted to pH 9.6 and incubated overnight at 4 • C. The microplates were then washed with PBS containing 0.1% Tween 20 (TPBS). The remaining protein-binding sites were blocked using 5% skim milk for 6 h at room temperature. After washing with PBS, 30 µg of protein samples were distributed into each well and incubated overnight at 4 • C. Each well was rinsed with TPBS and then incubated with primary antibodies diluted in PBS overnight at 4 • C (Table S2). After washing, peroxidase-conjugated secondary antibodies (Vector Laboratories, Newark, CA, USA) was loaded for 4 h at room temperature. Tetramethylbenzidine solution was added, followed by incubation for 15-20 min at room temperature. The stop solution that was used was 2 N sulfuric acid. The optical density was measured at a wavelength of 450 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).

Western Blotting
Equal amounts of proteins were separated on 8-12% polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA) by a power station (ATTO). After blocking using 5% skim milk and washing with Tris-buffered saline containing 0.1% Tween 20 (TTBS), we incubated the membranes with primary antibodies (Table S2) for 15 h at 4 • C and then washed them with TTBS. Next, the membranes were incubated with peroxidase-conjugated secondary antibodies (Vector Laboratories) at room temperature for 1 h and rinsed with TTBS. Subsequently, an enhanced chemiluminescence detection reagent (Cytiva TM , Seoul, Republic of Korea) and imaging system (ChemiDoc; Bio-rad, Hercules, CA, USA) were used to visualize the immunoreactive proteins on the membrane.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
The qRT-PCR reagent mixture was prepared by mixing 1 µg of synthesized cDNA, SYBR Green reagent (Takara), and 10 pmol of primer (Table S3). This mixture was added to a 384-well multi-plate and analyzed with a CFX386 Touch Real-Time PCR System (Bio-Rad).

Immunohistochemistry
The sectioned slides were passed through a series of xylene and ethanol solutions (100%, 90%, 80%, 70%) to remove the paraffin and then hydrated with distilled water and PBS. In order to reduce nonspecific binding, the slides were incubated with normal serum. The blocked slides were incubated with primary antibody (Table S2) for 15 h at 4 • C and 1 h at room temperature. The slides were then rinsed with PBS and incubated with biotinylated secondary antibodies (Vector Laboratories) for 1 h at room temperature. The slides were again rinsed with PBS and then incubated with ABC reagent (Vector Laboratories) according to the manufacturer's instructions. After washing with PBS, the slides were developed for 5 min using a 3,3 -diaminobenzidine tetrahydrochloride hydrate (DAB; Sigma-Aldrich). Then, the slides were washed with PBS, followed by distilled water, and counterstained with hematoxylin solution (DAKO, Glostrup Kommune, Denmark). After the slides were washed, they were dehydrated with absolute alcohol and mounted using xylene and dibutyl phthalate in xylene (DPX; Sigma-Aldrich). Images of the stained slides were acquired with an optical microscope (Olympus Optical Co., Tokyo, Japan), and the intensity was analyzed using ImageJ software (NIH, Bethesda, MD, USA). After deparaffinization, skin tissues were incubated in 0.5% periodic acid (BBC Biochemical, McKinney, TX, USA) for 5 min and rinsed with distilled water. Then, they were incubated in Schiff's reagent for 15 min and rinsed with running tap water. After they were incubated with hematoxylin for 2 min, the samples were rinsed with distilled water, dehydrated, mounted with DPX mount solution (Sigma-Aldrich), and observed under an optical microscope (Olympus Optical Co.) equipped with a slide scanner (Motic, Vancouver, British Columbia, Canada). All images were analyzed for collagen fiber density using ImageJ software (NIH).

Masson's Trichrome Staining
After deparaffinization, skin tissues were incubated in Bouin solution (Scytek Laboratories, West Logan, UT, USA) at 60 • C for 1 h and rinsed with distilled water. The sections were then placed in a working solution of iron hematoxylin (Scytek Laboratories) for 5 min, Biebrich scarlet acid fuchsin solution (Scytek Laboratories) for 5 min, phosphomolybdicphosphotungstic acid solution (Scytek Laboratories) for 12 min, and aniline blue solution (Scytek Laboratories) for 3 min. The stained slides were mounted with DPX mount solution (Sigma-Aldrich) and observed under an optical microscope (Olympus Optical Co.) equipped with a slide scanner (Motic). All images were analyzed for collagen fiber density using ImageJ software (NIH).
The fixed samples were embedded using a Poly/Bed 812 kit (Polysciences, Warrington, PA, USA) and polymerized in an electron microscope oven (DOSAKA, Katsumi, Japan) at 65 • C for 12 h. The block was equipped with a diamond knife in an ultramicrotome, cut into 200 nm sections, and stained with toluidine blue for optical microscopy.
The region of interest was then cut into 80 nm sections using the ultramicrotome, placed on copper grids, double stained with 3% uranyl acetate for 30 min and 3% lead citrate for 7 min, and observed by TEM (JEOL, Tokyo, Japan) equipped with a Megaview III CCD camera (Olympus Optical Co.) at an acceleration voltage of 80 kV.

Statistical Analysis
All results are presented as the means ± standard deviations, and all statistical analyses were performed using SPSS version 22 (IBM Corporation; Armonk, NY, USA). Statistical significance was determined by the Kruskal-Wallis test for comparisons of each group, followed by a post hoc Mann-Whitney U test. In this study, groups marked with different letters indicate significant intergroup differences. *

RA Decreased Oxidative Stress in H 2 O 2 -Treated Keratinocytes and Aged Animal Skin
First, we evaluated whether RA decreased oxidative stress in the cellular senescence model and aged animal skin. Since the H 2 O 2 -induced cellular senescence model is the most widely used in vitro aging model [43], we treated human keratinocytes with H 2 O 2 ( Figure S1A).
After treating keratinocytes with H 2 O 2 , PBS, AA, NA, and RA were administered ( Figure S1A). The oxidative stress alleviation effect of RA was compared with that of PBS, AA, and NA. NADPH oxidases are one of the primary ROS-generating enzymes [44].
NADPH oxidase activity was increased by treatment with H 2 O 2 . It was decreased by the administration of AA, NA, and RA. The most prominent decrease was observed in the RA-treated keratinocytes ( Figure 1A). SOD activity was decreased by treatment with H 2 O 2 . It was increased by the administration of AA, NA, and RA. The most prominent increase was observed in the RA-treated keratinocytes ( Figure 1B). 8-OHdG is a widely used marker of oxidative damage to DNA [45]. The expression of 8-OHdG was increased by treatment with H 2 O 2. It was decreased by the administration of AA, NA, and RA. The most prominent decrease was observed in the RA-treated keratinocytes ( Figure 1C). mtDNA damage was significantly increased by H 2 O 2 treatment. It was decreased by the administration of AA, NA, and RA. The most prominent decrease was observed in the RA-treated keratinocytes ( Figure 1D).
RA was injected two times into aged animal skin with an MTS every two weeks ( Figure S1B). The NADPH oxidase activity in aged animal skin was higher than that in young animal skin. It was decreased by RA injection ( Figure 1E).
The SOD level, which was evaluated with western blotting, decreased in aged skin compared with young skin. It was increased by RA injection ( Figure 1F,G).
The level of mtDNA damage in aged skin was higher than that in young skin, and it was decreased by RA injection. The 8-OHdG level in aged skin was higher than that in young skin, and it was decreased by RA injection (Figure 1H,I).
The findings indicated that RA decreased oxidative stress and oxidative stress-induced DNA damage.

RA Decreased Mitochondrial Dysfunction and Cellular Senescence in H 2 O 2 -Treated Keratinocytes and Aged Skin
PGC-1α, which is an essential controller of mitochondrial biogenesis [11], was decreased by treatment with H 2 O 2 . It was increased by the administration of AA, NA, and RA. The most prominent increase was observed in the RA-treated keratinocytes (Figure 2A). compared with young skin. It was increased by RA injection ( Figure 1F,G).
The level of mtDNA damage in aged skin was higher than that in young skin, and it was decreased by RA injection. The 8-OHdG level in aged skin was higher than that in young skin, and it was decreased by RA injection (Figure 1H,I).

RA Decreased Mitochondrial Dysfunction and Cellular Senescence in H2O2-Treated Keratinocytes and Aged Skin
PGC-1α, which is an essential controller of mitochondrial biogenesis [11], was decreased by treatment with H2O2. It was increased by the administration of AA, NA, and RA. The most prominent increase was observed in the RA-treated keratinocytes ( Figure  2A). Cytochrome c oxidase (COX)1 and succinate dehydrogenase complex, and flavoprotein subunit A (SDHA) are also mitochondrial biogenesis markers [46]. COX1 and SDHA levels were decreased by treatment with H 2 O 2 and increased by administration of AA, NA, and RA. The most prominent increase was observed in the RA-treated keratinocytes ( Figure 2B,C).
The levels of the mitochondrial fission markers DRP1 and FIS1 were decreased by treatment with H 2 O 2 and increased by administration of AA, NA, and RA. The most prominent increase was observed in the RA-treated keratinocytes ( Figure 2D,E). The levels of the mitochondrial fusion markers OPA1 and MFN2 were increased by treatment with H 2 O 2 and decreased by administration of AA, NA, and RA. The most prominent decrease was observed in the RA-treated keratinocytes ( Figure 2F,G).
The levels of the cellular senescence markers P21 and P16 [47] were increased by treatment with H 2 O 2 and decreased by administration of AA, NA, and RA. The most prominent decrease was observed in the RA-treated keratinocytes ( Figure 2H,I).
The levels of the markers of mitochondrial biogenesis, PGC-1α, COX1, and SDHA, were decreased in aged skin and were increased by RA injection (Figure 3A-D).  The protein expression levels of DRP1 and FIS1 were decreased in aged skin and increased by RA injection (Figure 3E-G).
The protein expression levels of OPA1 and MFN2 were increased in aged skin and decreased by RA injection (Figure 3E,H,I).
The expression of P21 and P16 was increased in aged skin and decreased by RA injection ( Figure 3J,K).

RA Decreased NF-Kb/AP-1 and MMP1/2/3/9 Expression in Aged Skin
The expression of NF-κB and AP-1 in aged skin was increased compared with that in young skin. However, these levels were decreased by RA injection (Figure 4A,B). The expression of NF-κB and AP-1 in aged skin was increased compared with that in young skin. However, these levels were decreased by RA injection (Figure 4A,B).
The expression of MMP1/2/3/9 in aged skin was increased compared with that in young skin. These levels were decreased by RA injection (Figure 4C-F).

RA Increased the Expression of Laminin and Nidogen and the BM Density
The expression of laminin and nidogen was significantly lower in aged skin than in young skin. These levels were increased by RA injection (Figure 5A-C).
The BM density was evaluated with PAS staining. The intensity of the pink color observed by PAS staining of the aged skin was lower than that of young skin; however, this intensity was increased by RA injection (Figure 5D,E).
The BM consists of three layers: the lamina lucida, lamina densa, and lamina fibroreticularis [48]. The lamina densa has a sheet-like structure. The lamina lucida exists between the lamina densa and the epithelial layer and forms hemidesmosomes, which are The expression of MMP1/2/3/9 in aged skin was increased compared with that in young skin. These levels were decreased by RA injection (Figure 4C-F).

RA Increased the Expression of Laminin and Nidogen and the BM Density
The expression of laminin and nidogen was significantly lower in aged skin than in young skin. These levels were increased by RA injection (Figure 5A-C).

RA Upregulated the Expression of TGF-Β, CTGF, and α-Smooth Muscle Actin (Α-SMA) and Collagen Fiber Accumulation in Aged Skin
In aged skin, the expression of TGF-β and CTGF was decreased compared with that in young skin. These levels were increased by RA injection (Figure 6A-C).
When fibroblasts are changed to activated myofibroblasts that express α-SMA, the expression of collagen type I and collagen type III increases [49,50].
The expressions of α-SMA, COL1A1, and COL3A1 were decreased in aged skin compared with young skin and increased by RA ( Figure 6A,D,E,F).
The collagen fiber density in the skin was evaluated with Masson`s trichrome staining. The collagen fiber density in aged skin was decreased compared with that in young skin, and this density was increased by RA injection (Figure 6G,H).
Skin elasticity was evaluated with API-100 ® (Aram Huvis Co., Ltd.). Skin elasticity change was decreased in aged skin and was increased by RA injection ( Figure 6I). The BM density was evaluated with PAS staining. The intensity of the pink color observed by PAS staining of the aged skin was lower than that of young skin; however, this intensity was increased by RA injection (Figure 5D,E).
The BM consists of three layers: the lamina lucida, lamina densa, and lamina fibroreticularis [48]. The lamina densa has a sheet-like structure. The lamina lucida exists between the lamina densa and the epithelial layer and forms hemidesmosomes, which are electrondense plaques [27]. It is known that photoaging induced disruption and duplication of the lamina densa [29].
The hemidesmosomes and lamina densa were observed by transmission electron microscopy. In aged skin, the lamina densa was more disrupted, and the number of hemidesmosomes was less than that in young skin. By RA injection, disruption of the lamina densa was improved, and the number of hemidesmosomes was increased ( Figure 5F).

RA Upregulated the Expression of TGF-B, CTGF, and α-Smooth Muscle Actin (A-SMA) and Collagen Fiber Accumulation in Aged Skin
In aged skin, the expression of TGF-β and CTGF was decreased compared with that in young skin. These levels were increased by RA injection (Figure 6A-C). (I) Skin elasticity was confirmed. Skin elasticity was decreased in aged mouse skin but increased by RA injection. **, p < 0.01, second bar vs. first bar; $$, p < 0.01, third bar vs. second bar (Mann-Whitney U test). α-SMA; αsmooth muscle actin, COL1A1; collagen type I alpha 1, COL3A1; collagen type III alpha 1, CTGF; connective tissue growth factor, ELISA; enzyme-linked immunosorbent assay, MW; molecular weight, TGF-β; transforming growth factor-β.
When fibroblasts are changed to activated myofibroblasts that express α-SMA, the expression of collagen type I and collagen type III increases [49,50].
The expressions of α-SMA, COL1A1, and COL3A1 were decreased in aged skin compared with young skin and increased by RA ( Figure 6A,D,E,F).
The collagen fiber density in the skin was evaluated with Masson's trichrome staining. The collagen fiber density in aged skin was decreased compared with that in young skin, and this density was increased by RA injection (Figure 6G,H).
Skin elasticity was evaluated with API-100 ® (Aram Huvis Co., Ltd.). Skin elasticity change was decreased in aged skin and was increased by RA injection ( Figure 6I).

Discussion
During intrinsic (chronological) aging, increasing levels of senescent epidermal or dermal cells lead to aggravation of DNA damage and mitochondrial dysfunction in neighboring cells [51]. Moreover, environmental factors such as UV radiation lead to increased deterioration of cellular senescence [52,53].
Collagen fibers play an essential role in maintaining skin elasticity by supporting the skin matrix [54,55]. Type I collagen is the main type of skin collagen and is mainly produced by fibroblasts in the dermis layer [56]. During aging, the ability of fibroblasts to synthesize collagen decreases by 1.0-1.5% each year, and decreasing collagen is accompanied by the formation of wrinkles [57,58].
During skin aging, changes in communication between keratinocytes and fibroblasts result in decreased levels of collagen fibers in the skin by both decreasing collagen synthesis and increasing collagen destruction [59][60][61]. Aged keratinocytes lead to more destruction of elastin fiber than young keratinocytes after UV exposure [62].
For skin rejuvenation, various effective formulas, including antioxidants, have been used via various delivery systems, such as topical creams, hypodermic needles, and microneedles [63]. Since topical creams only spread following the skin surface, the penetrating amount of drug is just 10-20% of the total amount of drug included in the cream [64]. Hypodermic needles can deliver almost 90-100% of the contained drug; however, they cause pain [65,66]. The delivery efficacy of microneedles is similar to that of hypodermic needles; however, microneedles do not cause pain [66]. Microneedles penetrate the stratum corneum and deliver drugs to the epidermis or upper dermal layer [66].
Since we delivered RA via MTS, we thought that RA could first affect keratinocytes in the epidermis and then keratinocyte-modulated fibroblasts in the dermis. Thus, we evaluated whether RA could decrease oxidative stress and mitochondrial dysfunction in H 2 O 2 -induced senescent keratinocytes.
We evaluated oxidative stress by measuring NADPH oxidase activity and SOD activity in H 2 O 2 -treated keratinocytes. After the H 2 O 2 treatment, NADPH oxidase activity was increased, and SOD activity was decreased. RA decreased NADPH oxidase activity and increased SOD activity. mtDNA damage and 8-OHdG levels, which are markers of oxidative damage to DNA, were increased by H 2 O 2 treatment. However, they were decreased by RA.
Similar to the result of the in vitro test, the NADPH oxidase activity of aged skin was increased compared with that of young skin. SOD expression was decreased in aged skin compared with young skin. After RA injection, NADPH oxidase activity decreased, and SOD expression was increased.
Endogenous ROS are mainly generated in the mitochondria since ROS are a byproduct of energy production [67,68]. During aging, chronic accumulation of ROS in the mitochondria leads to mutations in mtDNA that cause mitochondrial dysfunction [69][70][71].
After H 2 O 2 treatment, the levels of mitochondrial biogenesis markers (PGC-1α, COX1, and SDHA) were decreased, and these levels were increased by RA administration. Mitochondrial fission, which was evaluated by measuring the expression of DRP1 and FIS1, was decreased by H 2 O 2 treatment. The levels of these markers were increased by RA. Mitochondrial fusion, which was evaluated by measuring the expression of OPA1 and MFN2, was increased by H 2 O 2 treatment and decreased by RA. The levels of the cellular senescence markers P21 and P16 were increased by H 2 O 2 and decreased by RA. It seemed that H 2 O 2 induced mitochondrial dysfunction and cellular senescence in keratinocytes, and this effect was reduced by RA.
These changes were also observed in aged animal skin. Mitochondrial biogenesis and fission were decreased in aged skin and increased by RA. In contrast, mitochondrial fusion was increased in aged skin and decreased by RA. Cellular senescence in aged skin, which was evaluated by measuring P21 and P16, also increased and was decreased by RA injection.
NF-κB/AP-1/MMPs, which are involved in one of the main pathways involved in ECM destruction during aging [4][5][6], were evaluated in aged skin. The expression of NF-κB and AP-1 was increased in aged skin and decreased by RA injection. MMP1/2/3/9 levels were increased in aged skin and decreased by RA injection.
MMPs destroy the BM as well as collagen fibers in the ECM of the dermis [72]. During aging, the levels of proteins that form the BM are decreased [73]. Proteins that form the structure of the BM, such as collagen XVII, are also involved in transmembrane signal transduction during keratinocyte differentiation [74]. Laminin is also involved in supporting structural stability as well as modulation of cellular proliferation, migration, and differentiation [74]. Since BM plays an essential role in skin homeostasis, controlling the levels of BM proteins that are decreased by aging has been considered a method for decreasing skin wrinkles [75].
Epidermal keratinocytes express laminin and nidogen and secrete those proteins into the dermal-epidermal junction [76]. Then, laminin and nidogen are assembled into the BM [76]. Thus, keratinocyte function for modulating the BM is also important, in addition to fibroblast function.
We hypothesized that senescent keratinocytes might affect the expression of BM proteins such as laminin and nidogen by increasing oxidative stress. Thus, we evaluated the expression of nidogen and laminin in aged skin. These levels were decreased in the aged skin and increased by RA injection. The BM density, which was evaluated with PAS staining, was also decreased in aged skin and increased by RA injection.
Since increased oxidative stress also leads to decreased collagen synthesis by decreasing TGF-β [77], we evaluated the levels of TGF-β and CTGF, which are involved in collagen synthesis. In aged skin, the levels of TGF-β and CTGF were decreased, and these levels were increased by RA injection. Moreover, the expression of COLI and COLIII and collagen density were decreased in aged skin and increased by RA injection. Skin elasticity, which was evaluated with API-100, was decreased in aged skin and increased by RA.

Conclusions
Our study showed that RA decreased oxidative stress and mtDNA injury, which eventually decreased mitochondrial dysfunction in aged skin. RA also decreased ECM destruction by decreasing the levels of NF-κB/AP-1/MMPs and increasing the levels of BM proteins such as nidogen and laminin. RA enhanced the collagen synthesis-related signaling pathway of TGF-β and CTGF. These modulations associated with RA treatment led to increased collagen fiber accumulation and skin elasticity in aged skin.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12030694/s1, Table S1: List of reagents required for RA. Table S2: List of antibodies for western blotting (WB), immunohistochemistry (IHC), and enzymelinked immunosorbent assay (ELISA); Table S3: List of primers used for quantitative real-time polymerase chain reaction; Figure S1: Schematic diagram of the in vitro (A) and in vivo (B) experiment in this study.